Project summary The innate immune system is our first line of defense against viruses, bacteria, and cancer cells. How the innate immune system regulates itself is poorly understood, but we do know that over-activation of innate immunity can cause autoimmune diseases (e.g. lupus and gout) and neurodegenerative diseases (e.g. multiple sclerosis). Worryingly, most current modulators of innate immunity, such as attenuated bacteria, alum crystals, and steroids, are broad, non-specific, and poorly characterized. Thus, while strategies to modulate innate immune defense mechanisms have strong therapeutic potential, such interventions require deep understanding of their molecular underpinnings and their resulting effects on human physiology. I propose to exploit my interdisciplinary training in chemical biology and innate immunology to drastically increase the cure rates for human cancers by expanding our molecular understanding of innate immunity, which has lagged behind the pack of current immunotherapeutic strategies for treating cancer. I am captivated by the recently discovered innate immune second messenger 2'3'-cGAMP, which is immediately synthesized in the cytosol upon viral infection to elicit downstream immune responses. During my postdoctoral research, my hypothesis that 2'3'-cGAMP has anti-cancer therapeutic potential led me to develop stable, degradation-resistant analogs that caused remarkable tumor shrinkage in mouse models and are currently being tested in clinical trials by a pharmaceutical company. To preemptively learn how to minimize the adverse effects of 2'3'-cGAMP analogs in humans, I propose to characterize how 2'3'-cGAMP is regulated, either by active transport or enzymatic inactivation. We will illuminate how 2'3'-cGAMP, a small molecule with two negative charges, crosses the cell membrane and why its dominant hydrolase (ENPP1) is extracellular even though 2'3'-cGAMP only functions in the cytosol. We will also develop tool compounds to pharmacologically manipulate 2'3'-cGAMP transport and metabolism and to test their physiological roles in real disease settings. By dramatically increasing our knowledge of key molecular mechanisms that control 2'3'-cGAMP compartmentalization and physiology, both in cell-culture models and in disease models, the proposed high-risk, high-reward investigations will set the stage for developing effective strategies to manipulate these mechanisms, thus revolutionizing our paradigm for cancer immunotherapy.